Method for the production of nitric esters of hydroxysteroid lactones or their glycosides

ABSTRACT

Method for the production of a nitric ester of a hydroxysteroid lactone or a nitric ester of a glycoside of a hydroxysteroid lactone, the nitric ester containing at least one of free primary and/or secondary hydroxy groups, comprising subjecting a nitric ester of a hydroxysteroid lactone or a nitric ester of a glycoside of a hydroxysteroid lactone, the nitric ester containing more than one of primary and/or secondary nitrate groups and being free of primary and secondary hydroxy groups, to reduction reaction conditions for said more than one of primary and/or secondary nitrate groups thereby to partially convert the nitrate groups to free hydroxy groups. Novel nitric esters thereby produced include cymarol-19-nitrate, 16epi-gitoxin-16nitrate, gitoxin-16-nitrate, digoxin-12-nitrate and helveticosol19-nitrate. The nitric esters are useful as drugs having an inotropic or involuntary muscle-relaxing effect.

United States Patent [191 Scheller et al.

[ METHOD FOR THE PRODUCTION OF NITRIC ESTERS OF HYDROXYSTEROID LACTONES OR THEIR GLYCOSIDES [75] Inventors: Frieder Scheller, Berlin; Rudolf Megges, Berlin-Buch; Frank Dittrich, Dresden; Kurt Repke; Hans Joachim Portius, both of Berlin-Buch; Gunter Kammann, Radebeul; Hans-Jorg Schmidt, Dresden; Knut-Olaf Haustein, Erfurt, all of Germany [73] Assignee: Veb Arzneimittelwerk Dresden,

Radebeul, Germany [22] Filed: June 11, 1973 [21] Appl. No.: 369,025

[30] Foreign Application Priority Data June [5, 1972 Germany 165405 [52] US. Cl 260/2105; 424/182 [5 1] Int. Cl. C07J 41/00 [58] Field of Search 260/2105, 235. 209 D [56] References Cited OTHER PUBLICATIONS Pigman, Ward, The Carbohydrates," Academic Press lnc., Publishers, New York, I957, p. l69. Smith, p.a.s.. Open Chain Nitrogen Compounds" Vol. ll, Benjamin. Inc., New York, I966, p. 489.

[ Oct. 7, 1975 Kaufman et al. J.A.C.S., Vol. 74. pp. 4997-500l (i952).

Merrow et al. J.A.C.S. Vol. 75, pp. 4259-4265 (1953). Pigman, Ward, The Carbohydrates," Acad. Press lnc., Publishers, New York, 1957, p. l70.

Primary Examinerlohnnie R. Brown Assistant Examiner-Cary B, Owens Attorney, Agent, or Firm-Nolte and Nolte [57] ABSTRACT Method for the production of a nitric ester of a hydroxysteroid lactone or a nitric ester of a glycoside of a hydroxysteroid lactone, the nitric ester containing at least one of free primary and/or secondary hydroxy groups, comprising subjecting a nitric ester of a hydroxysteroid lactone or a nitric ester of a glycoside of a hydroxysteroid lactone, the nitric ester containing more than one of primary and/or secondary nitrate groups and being free of primary and secondary hy droxy groups, to reduction reaction conditions for said more than one of primary and/or secondary nitrate groups thereby to partially convert the nitrate groups to free hydroxy groups. Novel nitric esters thereby produced include cymarol-l9-nitrate, l6epi-gitoxinl6-nitrate, gitoxin-l6-nitrate, digoxin-lZ-nitrate and helveticosol-19-nitrate. The nitric esters are useful as drugs having an inotropic or involuntary musclerelaxing effect.

1 Claim, No Drawings METHOD FOR THE PRODUCTION OF NITRIC ESTERS OF HYDROXYSTEROID LACTONES OR THEIR GLYCOSIDES This invention relates to a method for the production of nitric esters of hydroxysteroid lactoncs or their glycosides which contain free primary and/or secondary OH-groups and to novel nitric esters thereby produced.

The presently known compounds of this type are produced by partial estcrification of hydroxysteroid lactones or their glycosides with nitric acid according to German Democratic Republic (East German) Pat. No. 81,106. But a great number of the many possible position-isomers cannot be produced according to this method, because the esterification with nitric acid occurs at the OH-groups successively depending on their reactivity.

The invention provides a method for the production of hitherto unobtainable or difficulty obtainable nitric esters of hydroxysteroid lactones or their glycosides which contain primary and/or secondary OH-groups.

According to the invention, primary and/or secondary nitrate groups of nitric esters of hydroxy lactones or their glycosides which contain no primary and/or secondary OH-groups, are partially converted into OH- groups by reduction. The reduction is effected electrochemically or by means of reducing substances, for example, sulfides, such as ammonium sulfide, thiols or sodium borohydride. The reduction is carried out according to the invention in a pH range between 3 and 10 and at temperatures between and 50C, preferably at room temperature.

Suitable are all solvents and solvent mixtures which dissolve the starting materials, do not react with them, and are not changed under the reaction conditions. Examples are lower alcohols, aliphatic acid amides, ethers, carboxylic acids, nitriles, amines, ketones, esters, if necessary in mixture with water.

Suitable as a conducting salt and buffer for the electrochemical reduction is, for example, tetra-N- butylammonium acetate (B Nac) or tris-(hydroxymethyl)-amino-methane --HCL buffer.

As starting materials of the method according to the invention are used the nitric esters which are easily obtainable according to German Democratic Republic Pat. No. 81,106 and which contains no primary and/or secondary OH-groups, for example, of gitoxigenin, lbepi-gitoxigenin, digoxigenin, strophanthidol, ouabagenin, digitoxin, gitoxin, digoxin, 17 a -hydroxydigitoxin, lanatoside A,B,C, desacetyllanatoside A,B,C, cymarol, helveticoside, helveticosol, convallatoxin, convallatoxol, ouabain, proscillaridin A, scillaren A, neriifolin, cerberoside, k-strophanthin-B, kstrophantholB, k-strophanthin'y, k-strophanthol'y, or

the corresponding l4-anhydroor 14,16-dianhydroor perhydro or 14,2l-epoxydihydro- (of Cardenolicles) or l9-carboxylic acid derivatives.

The reductiveconversion of nitric ester groups into The nitric esters produced according to the invention are biologically active and can be used as drugs with an inotropic or involuntary muscle-relaxing effect, also as valuable intermediate products for other syntheses.

The invention will be described below more fully on the basis of examples without being limited by them in any way.

EXAMPLE 1 300 mg. cymarol dinitrate are dissolved in 150 ml. isopropanol and 50 ml. 2N B Nac aqueous solution having a pH of 7 are added. The electrolysis is carried out at room temperature and a constant cathode potential of 650 mV between a mercury cathode and a platinum anode until 113 Faraday have flowed. The anode is separated from the cathode space by a glass dia' phragm. As a reference electrode is used as a saturated calomel electrode. The electrolyte is stirred and the oxygen is expelled with purified nitrogen. After dilution with water, the solution is extracted with chloroform. The extracted crude product contains approximately 90% cymarol-l9-nitrate which is obtained pure by chromatography (silica gel as adsorbent; ethyl acetate -ethanol-chloroform solvent mixture in the volume proportions 67.5 7.5 2 25) (184 mg. yield).

Thin-layer chromotography: silica gel G, 0.3 mm. coat thickness, solvent see above, two stage development.

Fluorescence color: orange (SbCl UV). mdinitrobenzenc reaction: positive (butenolidc ring).

Reduction (zinc, acetic acid, dioxane) yields cymarol.

UV (ethanol): max. 214 nm. 1R: -ONO 1275, 1625 cm, lactone-CO, 1730 cm. Acid hydrolysis yields strophanthidol -l9-nitrate', R,,,,,,,,,.,,,,,,,,-,,,,, 245 (chloroform 5% ethanol, two-stage development).

EXAMPLE 2 200 mg. cymarol dinitrate are dissolved in 200 ml. dioxane and 35 ml. 1N aqueous tris-(hydroxymethyl)- aminomethane-HCI buffer solution having a pH of 9 are added. With cooling to 0C, the procedure of Example is followed. 153 mg. cymarol-l9nitrate are obtained.

EXAMPLE 3 mg. cymarol dinitrate are dissolved in 40 ml. dimethyl formamide, 10 ml. aqueous 2N B Nac solution having a pH of 5 are added and the solution is reduced at 40C electrolytically as in Example 1. 59 mg. cymaroll 9-nitrate are obtained.

EXAMPLE 4 250 mg. 16 epi-gitoxin pentanitrate are dissolved in 450 ml. isopropanol and 50 ml. of an aqueous solution of 2N B Nac having a pH of 7 are added. The electrolysis is carried out with the same apparatus as in Example 1 at a potential of 800 mV. until 240 Faraday have flowed. The extracted steroid mixture contains about lfiepigitoxin l 6-nitrate which is obtained pure by chromatography on silica gel (yield, 142 mg).

ttir'ul-uilotrln 220 Thin layer chromatography: chloroform 10% by volume ethanol, two-stage development.

Fluorescence color: blue. m-dinitrobenzene reaction: positive (hutenolide ring).

Diphenylamine-UVreaction: positive (nitrate). Xanthydrol reaction: positive (digitoxose). Acid hydrolysis yields l6epi-gitoxigeninl 6-nitrate.

250. Thin layer chromotograttir-ukuilruim'uin m phy: as above.

Fluorescence color: blue. m-dinitrobenzcne-, diphenylamine -UV-reaction: positive. Xanthydrol reaction:

negative.

EXAMPLE mg. gitoxin pentanitrate are dissolved in 500 ml. isopropanol and 80 ml. aqueous 2N B Nac solution are added. The electrolysis is effected according to Example 4. The extracted steroid mixture contains gitoxinl6-nitrate, which is obtained pure by chromatography (yield, 8 mg.).

Thin layer chromatography: as in Example 4, but one-stage development.

Fluorescence color: blue m-dinitrobenzene reaction. diphenylamine-UV- reaction. xanthydrol reaction: positive.

EXAMPLE 6 100 mg. digoxin pentanitrate are dissolved in 300 ml. ethanol and 50 ml. aqueous 2N B Nac solution are added. The electrolysis is effected as in Example 4. The extracted steroid mixture contains digoxin-l2-nitrate which is obtained pure by chromatography on silica gel (59 mg.

Thin layer chromatography: as in Example 4 Fluorescence color: blue m-dinitrobenzcne reaction. diphenylamine-UV- reaction. xanthydrol reaction: positive.

EXAMPLE 7 EXAMPLE 8 I33 mg. proscillaridin trinitrate in 50 ml. ethanol is mixed with 1.89 g. cysteine hydrochloride in 30 ml. water and 40 ml. tris-(hydroxymethyl)-aminomethane-HCl buffer (pH 9) and the resultant solution is left standing under nitrogen for 8 days at room temperature. After dilution with water, the solution is extracted with chloroform. The chromatographic separation of the extracted steroid mixture yields the following nitrates:

diphenylamine-UV- "proscillaridin trinitrutc ltll) fluorescent yield.

color /1 l 72 brown-red 6 ill 25 7 Thin layer chromatography: chloroform-ethyl acetatehutanol-:19:1 parts by volume; developing time: 1 hour.

l-lll yield proscillaridin on reduction.

EXAMPLE 9 124 mg. ouabagenin tetranitrate are treated for 6 days as in Example 8. The following nitrates are obtained:

"ouabagenin tetranitrate 100 yield. 9!

I 80 27 ll 62 23 Thin layer chromatography: ethyl acetate-ethanol chloroform 67.5 7.5 25, two-stage development.

m-dinitrobenzene reaction: I. ll: positive.

EXAMPLE IO 96 mg. l6epi-gitoxigen dinitrate are treated as in Ex ample 8. After 7 days l6epi-gitoxigenin nitrate is obtained.

ltivpi-uilmrlm'nin (Iinllrulv 11H) Fluorescence color: blue Thin layer chromatography: as in Example 9, onestage development.

EXAMPLE I I To the solution of 100 mg. digoxin pentanitrate in 50 ml. alcohol, 370 mg. thioacetamide are added in 10 ml. concentrated ammonia and the mixture is left standing for 24 hours. Preparation according to Example 8 yields 3 nitrates:

"digoxin pentanitrate IUO fluorescence color yield, "/r

l 92 blue-grey 3l ll 87 33 Ill 4| 9 Thin layer chromatography: CHCl 5% ethyl alcohol by volume, two-stage development.

EXAMPLE 12 300 mg. digoxin pentanitrate is dissolved in 90 ml. alcohol, to which 940 mg. sodium borohydride in 10 ml. water as well as 36 mg. nickel nitrate in l ml. water are added, and is left standing for 30 minutes. Preparation as in Example 8 yields 2 digoxin nitrates:

"digoxin pentanitrate fluorescence color yield. 2

I 89 blue-grey 35 ll 82 49 Thin layer chromatography: CHCL; \5'7r ethyl ulcocymar0l-l9-nitratc, l6epi-git0xin-lfi-nitratc gitoxinhol by volume. l6 minute. digoxinl2-nitratc and hclvelicosol-IQ- What is claimed is: nitrate.

1. A compound selected from the group consisting of 

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF CYMARO-19-NITRITE, 16-EPI-GITO XIN-16-NITRATE, GITOXIN-16 -NITRATE DIOXIN-12-NITRATE AND HELVETICOSOL-19-NITRATE. 